Cyclic AMP binds to its receptor protein CRP, and induces a conformational change (allosteric transition) in the protein. Cyclic AMP.CRP complex, and not free CRP, binds at or near many promoters of E. coli in a sequence- specific way and either represses or activates transcription. We are investigating the structural changes in CRP, induced by cAMP, that are required to make the former proficient for gene regulation and the points of contact between cAMP.CRP complex and RNA polymerase needed to modulate transcription. We have isolated and characterized several classes of mutations in the crp gene, which identify the amino acids participating in the cyclic AMP-induced allosteric shift in the protein. Various substitutions for Aspl38 located at the apex of the hinge connecting the cAMP-binding domain to the DNA-binding domain of CRP cause biochemically distinct phenotypes. For example, Alal38 is defective in the cAMP-induced allosteric change, Asn138 functions in the absence of cAMP and Phe138 is defective for DNA binding. Thus, the hinge connecting the two domains of CRP is a key area for the transmission of the cAMP-induced allosteric change. In addition, this region is directly involved in DNA interaction. We are also studying the mechanism by which CRP activates transcription. We are analyzing this problem genetically by looking for CRP or RNA polymerase mutants that have lost the ability to interact with each other. In this regard, we have isolated mutations of E. coli which co-transduce with the beta and beta' genes encoding two RNA polymerase subunits. These mutations are being analyzed now. The mechanism by which a DNA-bound CRP communicates with RNA polymerase to activate it is also being studied. This is monitored by in vitro transcription using DNA templates with altered location or orientation of the CRP binding site with respect to the promoter or with a single stranded gap introduced between the CRP binding site and the promoter. We have found that OxyR, another transcriptional activator, is active only under non-reducing conditions.